Project Summary Approximately 10% of the world's adult population has chronic kidney disease (CKD) for which there are very few effective preventive or stabilizing therapeutic options. In addition, 30% of newly developed drugs are not advanced because of nephrotoxicity. We have developed efficient directed differentiation protocols to generate nephron progenitor cells (NPCs) and 3D kidney organoids from human pluripotent stem cells (hPSCs). At present, however, there are no effective platforms that integrate these kidney cells and vascularized organoids within microphysiological systems in vitro to develop effective kidney models for interrogation of nephrotoxicity and drug efficacy. Our proposal unites expertise in kidney organoids and disease, microphysiological systems, and bioprinting led by three experienced investigators (Bonventre, Lee and Lewis) in a unique effort to create these needed model platforms. In Specific Aim 1 we will develop efficient processes to direct differentiation of hiPSCs into kidney podocytes, tubular epithelial cells, and endothelial cells endowed with differentiated features for integration into microphysiological analysis platforms (MAP) and bioprinted structures. We create genetic models of disease and reporter lines that signal differentiation characteristics to optimize differentiation protocols and to monitor physiological parameters. In Specific Aim 2 we will design, construct, and characterize an integrated kidney MAP to evaluate the function of hPSC-derived kidney podocytes, endothelial and epithelial cells as well as kidney organoids. We will also use this platform to create a model of a glomerulus that will have differentiated podocytes on an extracellular matrix (to mimic the glomerular basement membrane) and hiPSC-derived endothelial cells on the other side of the basement membrane. The MAP will be optimized to interrogate basic kidney biology and pathobiology of both non-genetic and genetic disease involving kidney cysts or podocyte injury and test responses to putative therapeutic agents. In Specific Aim 3 we will bioprint a 3D kidney model that contains convoluted proximal tubules, pericytes and endothelial-lined vascular structures with controlled, physiologically relevant system. Modeled tubules and vasculature will be perfused through a open lumens. The ECM composition will be optimized to support confluent epithelialization using proximal and distal tubule cells, podocytes, and endothelial cells derived from hiPSCs. We will characterize polarized drug uptake, toxicity, and vectorial transport through the interstitium (ECM) as well as cell-cell interactions among the epithelial cells, interstitium and endothelium-lined channels to create and validate vascularized kidney models composed of cells derived from healthy and patients with cystic disease that affects the tubule and glomerular disease that affects the podocyte. Our program, with well-established milestones, will result in novel models to test kidney toxicity and drug efficacy.